The TLR7 agonist imiquimod enhances the anti-melanoma effects of a recombinant Listeria monocytogenes vaccine.
ABSTRACT Activation of innate immune cells through TLR triggers immunomodulating events that enhance cell-mediated immunity, raising the possibility that ligands to these receptors might act as adjuvants in conjunction with T cell activating vaccines. In this report, topical imiquimod, a synthetic TLR7 agonist, significantly enhanced the protective antitumor effects of a live, recombinant listeria vaccine against murine melanoma. This tumor protective effect was not dependent on direct application to the tumor and was associated with an increase in tumor-associated and splenic dendritic cells. Additionally, the combination of imiquimod treatment with prior vaccination led to development of localized vitiligo. These findings indicate that activation of the innate immune system with TLR ligands stimulates dendritic cell activity resulting in a bypass of peripheral tolerance and enhanced antitumor activity. The results of these studies have broad implications for future designs of immunotherapeutic vaccines against tumors and the treatment of metastatic melanoma.
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ABSTRACT: For over a century, inactivated or attenuated bacteria have been employed in the clinic as immunotherapies to treat cancer, starting with the Coley's vaccines in the 19th century and leading to the currently approved bacillus Calmette-Guérin vaccine for bladder cancer. While effective, the inflammation induced by these therapies is transient and not designed to induce long-lasting tumor-specific cytolytic T lymphocyte (CTL) responses that have proven so adept at eradicating tumors. Therefore, in order to maintain the benefits of bacteria-induced acute inflammation but gain long-lasting anti-tumor immunity, many groups have constructed recombinant bacteria expressing tumor-associated antigens (TAAs) for the purpose of activating tumor-specific CTLs. One bacterium has proven particularly adept at inducing powerful anti-tumor immunity, Listeria monocytogenes (Lm). Lm is a gram-positive bacterium that selectively infects antigen-presenting cells wherein it is able to efficiently deliver tumor antigens to both the MHC Class I and II antigen presentation pathways for activation of tumor-targeting CTL-mediated immunity. Lm is a versatile bacterial vector as evidenced by its ability to induce therapeutic immunity against a wide-array of TAAs and specifically infect and kill tumor cells directly. It is for these reasons, among others, that Lm-based immunotherapies have delivered impressive therapeutic efficacy in preclinical models of cancer for two decades and are now showing promise clinically. In this review, we will provide an overview of the history leading up to the development of current Lm-based immunotherapies, the advantages and mechanisms of Lm as a therapeutic vaccine vector, the preclinical experience with Lm-based immunotherapies targeting a number of malignancies, and the recent findings from clinical trials along with concluding remarks on the future of Lm-based tumor immunotherapies.Frontiers in Cellular and Infection Microbiology 01/2014; 4:51. · 2.62 Impact Factor
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ABSTRACT: Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR) ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.Journal of drug delivery. 01/2013; 2013:516749.
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ABSTRACT: Imiquimod and resiquimod represent Toll-like receptor (TLR) 7 and 8 agonists, which emerged as attractive candidates for tumor therapy. To elucidate immune cells, which mainly contribute to TLR7/8-mediated antitumoral activity, we investigated the impact of imiquimod and resiquimod on native human 6-sulfo LacNAc (slan) dendritic cells (DCs). We found that both TLR7/8 agonists significantly improve the release of various proinflammatory cytokines by slanDCs and promote their tumor-directed cytotoxic activity. Furthermore, resiquimod efficiently augmented the ability of slanDCs to stimulate T cells and natural killer cells. These results indicate that imiquimod and resiquimod trigger various immunostimulatory properties of slanDCs, which may contribute to their antitumor effects.Cancer letters 02/2013; · 5.02 Impact Factor
The TLR7 Agonist Imiquimod Enhances the Anti-Melanoma
Effects of a Recombinant Listeria monocytogenes Vaccine1
Noah Craft,2,3*†Kevin W. Bruhn,2†Bidong D. Nguyen,†Robert Prins,†‡Jia Wei Lin,‡
Linda M. Liau,‡§and Jeffery F. Miller†¶
Activation of innate immune cells through TLR triggers immunomodulating events that enhance cell-mediated immunity, raising
the possibility that ligands to these receptors might act as adjuvants in conjunction with T cell activating vaccines. In this report,
topical imiquimod, a synthetic TLR7 agonist, significantly enhanced the protective antitumor effects of a live, recombinant listeria
vaccine against murine melanoma. This tumor protective effect was not dependent on direct application to the tumor and was
associated with an increase in tumor-associated and splenic dendritic cells. Additionally, the combination of imiquimod treatment
with prior vaccination led to development of localized vitiligo. These findings indicate that activation of the innate immune system
with TLR ligands stimulates dendritic cell activity resulting in a bypass of peripheral tolerance and enhanced antitumor activity.
The results of these studies have broad implications for future designs of immunotherapeutic vaccines against tumors and the
treatment of metastatic melanoma. The Journal of Immunology, 2005, 175: 1983–1990.
both tumors and normal tissues, are commonly the chosen targets.
Therapies for melanoma in both animal and human studies have
included the Ags MART-1, gp100, tyrosinase, tyrosinase-related
protein (TRP)4-1, and TRP-2 (2–6). Overcoming self tolerance is
central to the success of this type of immunotherapy because the
target Ags represent immunologic self. The possibility for success
is suggested by prior evidence showing that T cells recognizing
melanocyte-specific differentiation Ags escape thymic deletion and
persist in the periphery (7). One therapeutic strategy is to enhance
the function of APCs such as dendritic cells (DCs) by providing
them with tumor Ags along with immunostimulatory signals that
induce maturation and augment the APC ability to activate T cells
CTL-mediated immunity can also be induced using live bacte-
rial vectors to stimulate the immune system and simultaneously
deliver Ags (11). Listeria monocytogenes is a Gram-positive, fac-
any cancer immunotherapies are centered on the acti-
vation of CD8 CTL that recognize specific tumor Ags
(1). Tissue specific differentiation Ags, expressed in
ultative, intracellular bacterium that is commonly used in studies
of cell-mediated immunity (12). Listeria infects many cell types,
including macrophages and other professional APCs (13, 14). Ags
expressed by Listeria can access both MHC class I and class II
processing pathways, and are presented to both CD8 and CD4 T
cells. Additionally, stimulation of TLR (15) on the surface
of APCs and the activation of internal pattern recognition mole-
cules such as nucleotide-binding oligomerization domain proteins
(16) may contribute to the immunostimulatory effects of Listeria.
Activation of these pathways leads to induction of inflammatory
cytokines such as IL-12, IFNs, and TNF-? (17–22) resulting in en-
hanced innate and adaptive responses and the promotion of cell-
mediated immunity to infection (23).
Our group and others have shown that L. monocytogenes can
induce protective cell-mediated immunity against heterologous
Ags. Recombinant L. monocytogenes (rLM) expressing viral pro-
teins can induce specific antiviral CD8 T cell responses (24–28)
and protect against virally induced tumors in animal models (29–
31). Additionally, the use of heterologous Ags as pseudo-tumor
Ags leads to rejection of tumors following rLM immunization (32,
33). Immunization with Listeria expressing the lymphocytic cho-
riomeningitis virus (LCMV) nucleoprotein (NP) led to s.c. rejec-
tion of a glioma cell line expressing heterologous NP (34). Inter-
estingly, animals clearing these tumors were subsequently immune
to rechallenge with glioma cells that did not express NP, suggest-
ing that epitope spreading had occurred (34). Recently, our group
demonstrated that rLM expressing the melanoma-associated Ag
TRP2 (LM-TRP2) was capable of inducing a TRP-2-specific CTL
response in BL/6 mice, and immunization with rLM expressing
TRP-2 led to partial protection from B16 challenge (35, 36). LM-
TRP2 could also be used therapeutically to treat established B16
tumors in naive mice (37). Strains that have been attenuated by
deletion of the virulence gene, actA, are also capable of generating
a protective immune response (37) and treatment of mice with
antibiotics 24 h following therapeutic immunization with nonat-
tenuated rLM did not inhibit the induction of antitumor immunity
(37). Additionally, strains that have been attenuated by deletion of
both the inlB and the actA virulence genes produce a robust and
*Division of Dermatology, Department of Medicine and Specialty Training and Ad-
vanced Research Program,†Department of Microbiology, Immunology, and Molec-
ular Genetics,‡Department of Surgery/Neurosurgery,§Jonsson Comprehensive Can-
cer Center, and¶Molecular Biology Institute, David Geffen School of Medicine,
University of California, Los Angeles, CA 90095
Received for publication December 21, 2004. Accepted for publication May 18, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a National Institutes of Health Grant RO1
CA84008-01 (to J.F.M.), a U.S. Public Health Service National Research Service
Award GM07104 (to K.W.B.), the Dermatology Foundation, Dermatologist Investi-
gator Research Fellowship and Clinical and Fundamental Immunology Training
Grant, and a National Institutes of Health/National Institute of Allergy and Infectious
Diseases Grant 5 T32 A1007126-27 (to N.C.).
2N.C. and K.W.B. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Noah Craft, Department of
Microbiology, Immunology, and Molecular Genetics, David Geffen School of Med-
icine, University of California, 43-319 Center for Health Sciences, 10833 LeConte
Avenue, Los Angeles, CA 90095-1747. E-mail address: email@example.com
4Abbreviations used in this paper: TRP, tyrosinase-related protein; DC, dendritic cell;
rLM, recombinant Listeria monocytogenes; NP, nucleoprotein; pDC, plasmacytoid
DC; LCMV, lymphocytic choriomeningitis virus.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
protective immune response with limited associated toxicity, thus
improving their potential utility as cancer vaccines in humans (38).
Imiquimod is an immunomodulatory compound in the imada-
zoquinoline family that displays both antiviral and antitumor ef-
fects (39, 40). Approved for the treatment of genital warts and
actinic keratoses, imiquimod has been used clinically for a wide
range of infectious and neoplastic skin disorders. A recent trial of
30 patients showed topical imiquimod to be effective in clearing
stage 0 melanoma (lentigo maligna) (41). The exact mechanisms
by which imiquimod mediates its effects in human melanoma are
currently unknown, although it is known to activate TLR7 (42).
Additionally, topical treatment with imiquimod induces a variety
of proinflammatory cytokines including IFN-?, TNF-?, and IL-12
(17–19) and facilitates the maturation and migratory capabilities of
DCs (43). Topical imiquimod treatment of mice with melanoma
led to accumulation of plasmacytoid DCs (pDCs) and partial clear-
ance of s.c. melanoma in the M3 DBA/2 mouse melanoma model
(44). Thus, we sought to determine the effectiveness of imiquimod
as an adjuvant during immunization with a live rLM vaccine ex-
pressing TRP-2. In this study, we found that the use of topical
imiquimod, when used in combination with the rLM vaccine ex-
pressing TRP-2, led to profound enhancement of the anti-
melanoma protective response and to localized vitiligo. These ef-
fects were not dependent on application of imiquimod directly to
the tumor, suggesting use of these compounds as vaccine adjuvants
may be relevant to metastatic melanoma immunotherapies as well.
Materials and Methods
Bacterial and mouse strains
Female C57BL/6 (H-2bMHC) mice were purchased from The Jackson
Laboratory and were between 6 and 10 wk of age and age-matched before
initiation of experiments. L. monocytogenes 10403S (45) (serotype 1/2a,
obtained from D. Hinrichs (Veterans Affairs Medical Center, Portland, OR)
via D. Portnoy (University of California, Berkeley, CA)) was the virulent
parental bacterial strain used for all recombinant constructs, and was grown
and maintained in brain-heart infusion broth or on agar plates with strep-
tomycin (200 ?g/ml) selection. Recombinant LM strains LM-NP and LM-
TRP2-NP expressing the LCMV NP396–404epitope (both strains) and
TRP2180–188(only LM-TRP2-NP) under control of the L. monocytogenes
hly listeria promoter and containing the hly signal sequence were created as
previously described (36). A defined, in-frame deletion was made in the
actA gene by allelic replacement as previously described (25) to create the
Immunization of mice
C57BL/6 mice (6 to 10 wk old) were inoculated in the tail vein with 0.1
LD50of each rLM strain in 200 ?l of PBS, using 28-gauge needles. Boost-
ing immunizations were given 2–3 wk later at a dose of 1.0 LD50. All
studies were conducted with the approval of the University of California,
Los Angeles Animal Research Committee (ARC).
Intracellular cytokine staining
Intracellular cytokine staining of splenocytes was performed as previously
described (36). Briefly, 1–2 ? 106splenocytes were stimulated in 96-well
flat-bottom plates with 1 ?M specific peptide or medium alone, plus brefel-
din A (BD Pharmingen) and 50 U/ml IL-2, for 6 h at 37°C in 5% CO2.
Cells were washed with staining buffer (PBS with 3% FBS and 0.09%
sodium azide), pretreated with anti-FcR Ab for 10 min, and then stained
with anti-CD4 FITC, anti-CD8 PE, and anti-CD44 CyChrome (BD Pharm-
ingen) at 1/100 final concentration, on ice for 20–30 min. Cells were then
permeabilized and fixed with Cytofix/CytoPerm (BD Pharmingen), then
stained for intracellular IFN-? with anti-IFN-? FITC or a FITC-labeled
isotype control mAb (1/100).
Tumor challenge and in vivo fluorescence imaging of tumors
The B16 murine melanoma cell line was obtained from the American Type
Culture Collection and maintained in DMEM supplemented with 10%
FCS, penicillin/streptomycin and L-glutamine. B16 cells stably expressing
firefly luciferase (B16-Fluc) were created as described elsewhere (35).
Growth rates of B16-Fluc both in vitro and in vivo were similar to those of
parental B16 cells. Before tumor challenge, B16-Fluc cells were grown in
supplemented DMEM, harvested, washed three times, and resuspended in
PBS. For s.c. tumors, mice were anesthetized, shaved on the flank, and
injected s.c. with 1 ? 104tumor cells in 50 ?l of PBS and 50 ?l of Matrigel
(BD Biosciences). For metastatic (lung) tumors, mice were immobilized
and injected i.v. with 1 ? 104tumor cells in 200 ?l of PBS into the tail
vein. Before imaging, mice were anesthetized with a mix of ketamine to
xylazine (4:1) in PBS, injected i.p. with 100 ?l of 30 mg/ml luciferase
substrate, D-luciferin (Xenogen) in PBS, and shaved over the injection area
(s.c.) or chest (lung metastases) to minimize the amount of light absorbed
by black fur. A cooled charge coupled device camera apparatus (IVIS;
Xenogen) was used to detect photon emission from tumor bearing mice
with an acquisition time of 2 min. Analysis of the images was performed
as previously described (46, 47) using Living Image software (Xenogen)
and Igor Image analysis software (Wave Metrics) by drawing regions of
interest over the tumor region and obtaining maximum values in photons/
second/cm2/steradian. Imiquimod (3M) was applied daily as a 5% cream to
shaved skin at the tumor site or to the flank. In experiments in which
imiquimod was applied to a distant site, tumors were placed s.c. on the
abdomen and imiquimod 5% cream was applied to the abdomen or to the
back. Control mice were treated with vehicle control.
All error bars represent SEM. Significant differences of tumor growth,
intracellular staining, splenic weight, and number of splenocytes were as-
sessed by Student’s t test. The difference between groups was considered
statistically significant when value p ? 0.05.
Immunohistochemical staining was performed as previously described
(48). Primary Abs used were against CD3? (145-2C11; BioDesign Inter-
national), CD4 (GK1.5; BioDesign International), CD8? (KT15; Bio-
Source International), or CD11c (HL3; BD Biosciences). The primary
mAb incubation step was followed by a biotinylated secondary mAb (Vec-
tor Laboratories) and developed with a diaminobenzidine or Nova Red
substrate kit (Vector Laboratories). Negative controls consisted of isotype-
matched rat or hamster IgG in lieu of the primary mAbs as listed.
Bacterial recovery assay and survival curve
Infected mice were sacrificed and liver and spleen were homogenized in
1% Triton X-100/PBS. Serial dilutions of homogenates were plated on
brain-heart infusion/streptomycin agar plates and colonies were counted
after growth at 37°C for 24 h. For survival curves, infected mice were
monitored daily for signs of systemic illness. Moribund animals were sac-
rificed according to ARC guidelines.
Topical imiquimod leads to a partial antitumor response
Imiquimod has recently been shown to be partially effective as a
therapy against melanoma in situ in humans (30). To determine
whether imiquimod is effective in treating B16 melanoma tumors,
we s.c. challenged mice with B16 cells expressing firefly luciferase
(B16-Fluc) and treated the site daily with topical 5% imiquimod
cream. Tumors were monitored by bioluminescent imaging, and
results shown in Fig. 1A represent tumor volume 2 wk after im-
plantation. In two separate experiments, a partial response was
noted, but the effects were variable and were not statistically sig-
nificant. However, histological examination of the tumor sites re-
vealed destruction of tumors in mice that received imiquimod
compared with animals that did not (Fig. 1B). A similar response
to lung metastatic tumors was also noted in mice challenged with
B16 cells i.v. and treated topically with imiquimod alone (data not
shown). These observations demonstrate that imiquimod treatment
alone induces partial and variable tumor destruction, both locally
and via systemic immunomodulation.
Topical imiquimod induces systemic immunomodulation
During experimental dissections, we noted marked splenomegaly
in imiquimod-treated mice, but normal spleens in naive animals or
animals with tumor alone. Previous studies have demonstrated that
imidazoquinolines can stimulate splenocyte proliferation ex vivo
1984 IMIQUIMOD POTENTIATES MELANOMA VACCINE
(49). Other studies have shown that imiquimod causes an increased
migratory capacity of DCs to draining lymph nodes (43). A more
recent study demonstrated that imiquimod induces splenomegaly
and an accumulation of “pDC-like” cells in the spleen (44). Thus,
we hypothesized that application of topical imiquimod is likely
responsible for producing the enlarged spleens. Daily treatment of
tumor bearing or naive mice with topical imiquimod caused a
marked and reproducible increase in splenic weight (Fig. 2A) and
total number of splenocytes (data not shown). The presence of
tumor did not affect the induced splenomegaly. Spleens were char-
acterized by a loss of germinal center architecture, a massive in-
crease in larger monocytic cells, and the appearance of many
multinucleate giant cells compared with spleens in controls (Fig.
2B). No significant changes were observed in the numbers of
CD8?or CD4?T cells, or NK cells (CD3?, NK1.1?) by FACS
analysis (Fig. 3A). There was a moderate increase in the number of
B220?cells. However, these B220?cells likely represent a subset
of pDC-like cells based on recent reports of imiquimod effects in
other mouse strains (44). Increases in splenic NK-T cells (CD3?,
NK1.1?), myeloid lineage cells (Gr-1?, CD11b?), and DCs
(CD11c?) were most pronounced (Fig. 3). The striking splenic
phenotype in imiquimod-treated animals accompanied by the sub-
stantive influx of immune cells demonstrates that imiquimod ap-
plied topically to the skin results in potent effects both systemically as
well as locally. Because DCs are professional APCs and are capable
of T cell activation, we hypothesized that imiquimod would enhance
the antitumor protection induced by an anti-melanoma vaccine. To
s.c. tumor challenge in naive mice. A, Groups of seven naive mice were
challenged with 1 ? 104B16-Fluc cells s.c. and treated daily with vehicle
control or with 5% imiquimod cream over the implantation site. Tumor
volume was monitored with real-time bioluminescent imaging as de-
scribed. Graph represents mean ? SE of tumor size at 2 wk and is repre-
sentative of other time points. Similar results were seen in a separate ex-
periment (not statistically significant). B, Mice received either nothing
(left) or topical imiquimod every day (right) to the tumor site. Loss of
tumor architecture and increased tumor necrosis are seen in tumors treated
with imiquimod as shown in representative frozen sections from day 18
tumors (H&E stained).
Imiquimod treatment leads to partial protection from B16
influx of DCs. A, Groups of four mice each with and without B16 tumors
were treated daily with topical imiquimod for 18 days. Spleens were har-
vested and weighed and mean ? SE is shown. Topically applied imi-
quimod led to a significant increase in the splenic weight in both the un-
challenged and tumor bearing mice. B, Groups of six mice each were
treated daily for 18 days with topical imiquimod in the absence of B16
tumors or rLM. Histological analysis of the spleens shows a loss of typical
germinal center architecture and a massive influx of macrophages and
monocytes as well as multinucleate giant cells. Staining with CD11c Ab
demonstrates the majority of these larger cells are DCs.
Topical imiquimod treatment induces splenomegaly and an
1985 The Journal of Immunology
test this hypothesis we used a recombinant L. monocytogenes vac-
cine expressing the melanoma Ag TRP-2.
Imiquimod treatment leads to increased susceptibility to
infection with Listeria
Infection of mice with rLM expressing the melanoma self-Ag
TRP-2 generates Ag-specific CD8?CTL and confers partial pro-
tection against B16 melanoma progression (36). We hypothesized
that the addition of imiquimod during vaccination would enhance
antitumor protection resulting from rLM immunization. To test
this hypothesis, we treated groups of C57BL/6 mice with topical
5% imiquimod cream for 3 days preceding bacterial challenge.
LM-TRP2-NP or the control strain (LM-NP) was used. Both
strains also express the immunodominant H-2Kbrestricted NP396–
404 epitope from LCMV as a readily measurable, internal, non-self
epitope control. Imiquimod was applied daily to the shaved flank
of mice for 3 days before vaccinating with either rLM strain in the
tail vein, and treatment was continued daily following vaccination.
Surprisingly, in comparison to untreated mice, 90% of imiquimod-
treated mice died 2–4 days following rLM injection (Fig. 4A). To
determine bacterial loads in imiquimod-treated animals, we
counted CFUs in the liver and spleen 48 h following injection (Fig.
4B). Topically applied imiquimod clearly inhibited the ability to
control a normally sublethal infection of LM-NP or LM-TRP2-NP.
To determine whether death was dependent upon bacterial growth
and spread, we repeated the experiment using a mutant Listeria
strain (?actA) that was unable to polymerize host cell actin and
spread from cell to cell (50). This attenuated strain did not cause
death in mice pretreated with imiquimod, when given at the same
dose as wild-type rLM (data not shown), suggesting that mice were
dying as a result of bacterial growth and spread, and not simply
due to exposure to the initial inoculum. Additionally, when LM-
TRP2-NP was inoculated at nonlethal doses in combination with
imiquimod, no augmentation of the CD8?T cell response was
noted (data not shown). Given the lethality of this combined treat-
ment and the apparent lack of augmentation of the T cell response,
we sought to determine the effects of imiquimod on tumor protec-
tion in the context of an already existing memory T cell
eloid and NKT cells. A, Immunostaining and FACS analysis demonstrated
no significant change in the number of CD4?or CD8?lymphocytes, or
NK cells after imiquimod treatment. There is a modest increase in the
number of B220?(likely B220?DCs) and NKT cells in the spleens of
imiquimod treated mice. B, In imiquimod-treated mice there is a marked
increase in the number of CD11b?and CD11c?DCs as well as Gr-1?
myeloid cells. Data represent individual mice (symbols) and the mean is
shown by thick bars. ?, p ? 0.05; ??, p ? 0.01.
Topical imiquimod treatment induces splenic influx of my-
mary Listeria infection. A, Groups of four to six mice each were treated
with nothing or with topical imiquimod daily for 4 days. Then, on day 0,
mice were infected with 0.1 LD50of various strains of rLM as indicated
and observed. Mice that received imiquimod treatment continued to receive
daily application of topical 5% imiquimod cream. Topical imiquimod led
to decreased survival after infection with virulent rLM strains LM-NP and
LM-TRP2-NP. B, Groups of six mice each were treated with nothing or
with topical imiquimod daily for 4 days. Mice were then infected with
LM-NP, spleens and livers were excised after 48 h, and bacterial counts
were determined per organ. Treatment with imiquimod was associated with
decreased ability to clear Listeria as shown by increased bacterial counts in
the organs from imiquimod-treated mice. Data are mean ? SE.
Imiquimod pretreatment increases the susceptibility to pri-
1986 IMIQUIMOD POTENTIATES MELANOMA VACCINE
Imiquimod enhances vaccine-induced anti-melanoma immunity
Prior studies in our laboratory demonstrated that immunization
with LM-TRP2-NP is capable of inducing a specific CTL response
against TRP-2 and that immunity is associated with protection
from challenge with B16 melanoma cells (36, 37). We hypothe-
sized that treatment of tumors with imiquimod in the context of
vaccine-induced memory T cells would enhance the antitumor re-
sponse. To determine the effects of imiquimod as a vaccine adju-
vant in this setting, we administered imiquimod to tumors in pre-
viously vaccinated mice. C57BL/6 mice were immunized and
boosted with LM-TRP2-NP or LM-NP. Two weeks later, mice
were s.c. challenged with B16-Fluc cells. Mice were then treated
with imiquimod 5% cream daily to the tumor implantation site.
The protection normally conferred by the LM-TRP2-NP vaccine
strain was significantly enhanced by the application of topical imi-
quimod (Fig. 5A). Imiquimod conferred only partial and variable
antitumor protection when applied to mice immunized with the
control LM-NP strain (Fig. 5A) or to naive mice (data not shown).
In several individual experiments the effects of imiquimod alone
were variable. In contrast, the combination of rLM immunization
and topical imiquimod led to profound and highly reproducible
tumor rejection in this model.
To determine whether the synergistic effects of imiquimod were
dependent on application directly to the tumor, we repeated these
experiments with imiquimod cream applied to a site distant from
the tumor. Mice that had imiquimod applied to a distant site were
almost as equally protected as mice that had imiquimod applied
directly to the tumor (Fig. 5B). This finding suggests that topically
applied imiquimod induces systemic antitumor immunity. Al-
though imiquimod clearly increased the efficiency of tumor rejec-
tion, it did not result in a quantitative increase in TRP-2 specific
CD8?T cells in the spleen (Fig. 5C).
To further probe the ability of imiquimod to enhance the sys-
temic effects of the rLM vaccine, we immunized and boosted mice
with either LM-TRP2-NP or LM-NP and then challenged the mice
2 wk later with i.v. delivered B16-Fluc cells. Imiquimod or pla-
cebo control cream was applied to the shaved flank daily. Mice
were sequentially imaged with bioluminescent imaging to monitor
tumor growth in the lungs. Mice that received either imiquimod
alone or vaccine alone were partially protected from lung tumor
development and resulting morbidity (Fig. 6A). However, mice
that received LM-TRP2-NP and topical imiquimod were pro-
foundly protected from lung tumor development (Fig. 6A) and
none had died by day 31 posttumor challenge. As was noted in the
spleen, mice treated with imiquimod showed an accumulation of
CD11c?DCs in lungs bearing tumors as measured by flow cy-
tometry (Fig. 6B). This finding was independent of prior immuni-
zation with LM-TRP2-NP.
To determine the long term outcome of the antitumor protection
caused by the combination of imiquimod and a pre-existing rLM-
induced anti-TRP-2 CTL response, we continued one experiment
with s.c. B16-Fluc challenge past the 3 wk when all other control
titumor immunity. A, C57BL/6 mice were immunized and boosted with
either LM-NP or LM-TRP2-NP and then challenged s.c. with 1 ? 104
B16-Fluc cells. Tumor fluorescence from B16-Fluc tumors was quantified
as described in Materials and Methods. Topical imiquimod applied to B16-
Fluc tumor sites resulted in somewhat decreased tumor growth at 2 wk in
mice immunized with control rLM (LM-NP) (p ? 0.13). Immunization
with rLM expressing TRP-2 (LM-TRP2-NP) consistently resulted in de-
creased tumor growth as shown previously (p ? 0.001). The effect of
topical imiquimod was synergistic with less variable and more profound
protection in the mice immunized with LM-TRP2-NP (p ? 0.00001).
Graph represents the mean of four separate experiments of four to eight
mice per group each compared with control growth at 2 wk (mean ? SE).
B, After primary and secondary immunization with LM-NP or LM-TRP2-
NP, mice were challenged s.c. on the abdomen with B16 tumors as de-
scribed. Daily application of 5% imiquimod cream led to significantly
Imiquimod treatment enhances protective rLM-induced an-
increased tumor protection when applied directly to the tumor or to the
back of the mouse away from the tumor. Graph is a representative exper-
iment with six mice per group (mean ? SE). C, Splenocytes were har-
vested from mice with or without B16 tumors and after vaccination with
LM-TRP2-NP, application of topical imiquimod, or both. Presence of in-
tracellular IFN-? secretion was determined after stimulation with various
peptide epitopes in vitro using flow cytometry. Percentage of IFN-?-pos-
itive cells is shown as a mean of four mice per group ? SE. Topical
imiquimod did not alter the percentage of CD8?Ag-specific epitopes in
either naive or vaccinated mice.
1987 The Journal of Immunology
groups had large tumors necessitating euthanasia. Surprisingly, by
6 wk, six of seven mice in the group that received LM-TRP2-NP
and imiquimod were still alive and four of the seven animals had
no detectable tumors. Lastly, the same four mice without tumors
also developed localized vitiligo over the site of tumor implanta-
tion and imiquimod application (Fig. 7). This finding had not been
observed in prior experiments with imiquimod or rLM vaccine
alone and indicates that although the effects of imiquimod can be
seen systemically, they are most potent in the skin under the ap-
plication site and clearly represent the ability to bypass peripheral
We have demonstrated the ability of the synthetic TLR7 agonist
imiquimod to potentiate the antitumor effects of a recombinant L.
monocytogenes vaccine against melanoma. Complete protection of
mice from the aggressive B16 murine melanoma is a challenge not
met by many therapies. This effect is most powerful locally, as
exemplified by the localized vitiligo where imiquimod was applied
and represents a clear breaking of peripheral tolerance. However,
the potentiation of prior vaccination is not dependent on applica-
tion directly to the tumor. This observation has broad implications
for immunotherapy against metastatic disease.
titumor immunity against metastatic melanoma. A, C57BL/6 mice were
immunized and boosted with rLM expressing the LCMV NP396–404
epitope (LM-NP) or the NP396–404epitope and the melanoma Ag TRP2180–
188 epitope (LM-TRP2-NP) and challenged i.v. with 1 ? 104B16-Fluc
cells into the tail vein. Tumor fluorescence from B16-Fluc tumors in the
lungs was quantified weekly as described in Materials and Methods. Top-
ical imiquimod was applied to the shaved flank daily and resulted in partial
and variable protection from B16 challenge in mice that received LM-NP.
Immunization with LM-TRP2-NP consistently resulted in partial antitumor
protection as shown previously. Graph represents the mean of four or five
mice for all days except day 31 at which the number of surviving mice is
indicated. B, Lungs from tumor bearing mice were excised and single cell
suspensions were made by passing through a nylon mesh. Immunostaining
with anti-CD11c Ab and FACS analysis was performed. Data are the per-
centage of CD11c?staining cells from groups of four mice each and are
the mean ? SE.
Imiquimod treatment enhances protective rLM-induced an-
juvant to rLM vaccine. C57BL/6 mice were immunized and boosted with
rLM expressing the LCMV NP396–404epitope (LM-NP) or the NP396–404
epitope and the melanoma Ag TRP2180–188epitope (LM-TRP2-NP) and
challenged s.c. with 1 ? 104B16-Fluc cells. Mice were then treated with
either 5% imiquimod or vehicle control cream daily for 3 wk. Mice that
received either the control vaccine strain or the vehicle control had to be
euthanized after 3 wk due to tumor burden. Six of seven mice that received
LM-TRP2-NP and imiquimod had no visible tumors at 3 wk. Four of seven
mice in this group developed localized vitiligo at the imiquimod applica-
tion site after 4 wk and still had not developed tumors. An example of a
control mouse is shown for comparison (A), and a mouse that was immu-
nized with LM-TRP2-NP and treated with imiquimod (B) is also presented.
Imiquimod induces localized vitiligo when used as an ad-
1988 IMIQUIMOD POTENTIATES MELANOMA VACCINE
One hypothesis to explain the synergistic effects of topical imi-
quimod with LM-TRP2-NP vaccination is that imiquimod treat-
ment results in an expanded population of vaccine-induced CD8?
Ag-specific CTLs. We did not observe a quantitative change in the
number of TRP-2-specific CD8?T cells in the spleen. However,
imiquimod treatment alone led to a significant DC response in both
the tumor and the spleen, but did not lead to reproducible antitu-
mor protection in the absence of a vaccine-induced memory T cell
population. An alternate hypothesis is that imiquimod-induced
DCs may enhance the vaccine-induced antitumor response by
stimulating the cytolytic function of vaccine-specific CD8?T cells
at the tumor site. This hypothesis is supported by the fact that
TLR7 agonists can synergize with CD40L to stimulate CD8 re-
sponses (51) and can uniquely induce IL-12 and TNF-? from
CD11c?, CD11b?, CD8?DCs (52). IL-12 pretreatment is known
to potentiate the antitumor effects of IFN-? in the B16 mouse
melanoma model (53). Lastly, in the setting of established toler-
ance, recent evidence suggests that persistent TLR signaling is
required for bypassing regulatory T cell-induced tolerance (54).
Indeed, after 3 wk of daily imiquimod application in the presence
of a pre-existing CTL response against the melanocyte-associated
Ag TRP-2, localized vitiligo was induced, clearly demonstrating a
break of peripheral tolerance to self-Ags.
The increase of DCs in the spleen and at the tumor site raises the
possibility that these cells are involved in imiquimod’s mechanism
of action. The molecular phenotypes of these cells have recently
been characterized (44), but their role in tumor clearance is still
unclear. Because the number of DCs is increased in animals that
have not received the rLM vaccine and are not protected from
tumor challenge, other factors are likely involved. We propose a
two-step model to explain our observations. First, LM-TRP2-NP
immunization leads to expansion of TRP-2-specific CD8?T cells
that home in on and partially destroy the tumor. Application of
imiquimod then leads to enhanced activation and migration of DCs
to the tumor site. These DCs are better able to process tumor debris
and present other tumor Ags to naive circulating T cells. This
phenomenon, termed “determinant spreading,” has been shown to
correlate highly with melanoma regression responses in human
vaccine trials (55). We have previously shown that vaccination
with rLM expressing a pseudo-tumor Ag can lead to functional
determinant spreading in a rat glioma model (56). The contribution
of determinant spreading to the augmented protection observed in
our model is currently being assessed.
The observation that imiquimod treatment leads to increased
susceptibility to bacterial infection is somewhat contrary to the
idea that TLR agonists can enhance DC maturation and function
(57). Indeed, imiquimod has been demonstrated to enhance clear-
ance of another intracellular organism, Leishmania, both in vivo
and in vitro in macrophages (58). However, although activated
DCs are adept at presenting Ag and inducing adaptive immune
responses, they are not as effective as macrophages at directly
killing intracellular bacteria (59, 60), and may be less susceptible
to T cell-mediated killing than other cell types (61, 62). Therefore,
the large numbers of DCs induced by imiquimod may serve as a
protected reservoir for Listeria to replicate undisturbed. Alterna-
tively, recent findings by our group (63) show that mice deficient
in type I IFN signaling are protected from Listeria-induced splenic
apoptosis. Additionally, type I IFNs induced by injection of poly
(I:C), a TLR3 agonist, led to increased susceptibility to Listeria
infection in wild-type mice, but not in mice defective in type I IFN
signaling. These findings suggest that imiquimod-induced suscep-
tibility to Listeria might be due to splenic apoptosis dependent
upon increased type I IFNs, which are known to be induced by
Although the natural ligand for TLR7 was recently determined
to be ssRNA (64), synthetic TLR7 and TLR8 agonists are also
potent TLR activators. In this study we demonstrate that a vaccine-
induced antitumor T cell response can be augmented systemically
by topical application of imiquimod. This novel finding provides
evidence that TLR activation can lead to enhanced systemic im-
munity and may lead to improved immunotherapies for metastatic
disease. Additionally, our observation that enhanced tumor pro-
tection is associated with an inability to clear an intracellular bac-
terial infection shows that the delicate balance between innate and
adaptive immunity will be an important consideration when using
these agents clinically, especially in immunocompromised pa-
tients. Further studies on the exact mechanisms of tumor protection
and bacterial susceptibility are warranted to assess the potential of
imiquimod for treating neoplastic disease.
The authors have no financial conflict of interest.
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